Apoptosis in mouse J774 macrophages-a
methodological study
By : Md. Hasan Imam
Main Supervisor: Olle Stendahl
Co-supervisor: Veronika Brodin Patcha ISRN: LiU-HU/MEDBIO/SP-A-2009/23-SE
Scientific Project Presented At the University of Linköping in Partial Fulfillment of The Requirements for the Degree of Master of Medical Bioscience
This project was performed at the division of Medical Microbiology Department of Clinical and Experimental Medicine
Linköping University. Sweden June, 2009
1. Abstract:
The approach of studying apoptosis induced by UV ray and actinomycin treatment was done to find a suitable positive control for that apoptosis study, with murine macrophage (J774 cell line). This scientific project was done in order to develop a method with this cell line in our laboratory condition for further apoptosis study in the presence of M.tuberculosis bacilli. It is well known that Mycobacterium tuberculosis primarily targets the macrophages and use them as a critical reservoir for the infection in the lung. Macrophages show several antimicrobial activities in order to limit the spread of intracellular infection during an attack by tuberculosis bacilli and apoptosis is one of the innate defense mechanism.
Our findings were important because it gives the necessary information’s regarding the possible sites where care is to be taken during working with this cell line (J774). We investigated the effect of UV exposure and actinomycin D treatment to find the suitable positive control for the study of apoptosis in J774 cells. Moreover, we studied effect of yeast particles on apoptosis and the phosphorylation of p38MAPK at two different time intervals in J774 cells. In order to quantify the percentage of apoptotic cells, we stained the cells by FITCAnnexin V, Propidium Iodide, and TMRE and evaluated the results by flow-cytometry.
Additionally, the phosphorylation of p38MAPK was assessed by western blot. The results indicated steady increase in apoptosis, measured by both annexin-V and TMRE 18 hours after UV exposure. Also we investigated phosphorylated p38MAPK after 18 hours from UV exposure (result not given) but no band was found. Actinomycin on the other hand, had no effect on the number of apoptotic cells and further study is needed to optimize its concentration. Non opsonised yeast did not induce any increase in annexin-V staining compared to the control, but did surprisingly induce a decrease in the mitochondrial membrane potential after 6hours, an effect that was diminished after 18 hours, here also we didn’t find any band of that phosphorylated p38MAPK during immunoblotting. Similar data were found with serum opsonised yeast particles. Maybe the beta-glucan of yeast particle was involved here by producing reactive oxygen species via NADPH oxidase and consequent mitochondrial membrane potential loss but we couldn’t draw any conclusion from our result about apoptosis occurring.
2. Popular summary
Our focus was to establish an experimental design of studying macrophage cell death in the presence of Tubercular bacterium, because this macrophage cell death is one of an immune mediated defense against M. tuberculosis. Two types of control was there to obtain validity of the experiment with this J774 murine derived macrophage cell line. The positive control was exposed to different stress conditions such as ultraviolet radiation and actinomycin (cytotoxic agent), which will give the idea that the system is working. This positive control will prove the comparative amount of cell death occurred by any other means. As a negative control we recommended only cell without any treatment which was thought to have cells with only spontaneous cell death.
We also used yeast particle both serum opsonised and non opsonised to observe the effect of the yeast beta-glukan on cell death. This serum opsonisation prepares the macrophage to engulf the yeast particle through an immune pathway.
The exposure of phosphatidyl serine (PS) on the outer surface of cell membrane and the dispersion of TMRE dye through out the cytosol during mitochondrial resting membrane potential loss are two symptoms of a dying cell we targeted. Fluorescent tagged Annxin-V which is very specific to that PS and the TMRE dye were used to stain the cell and were measured by flocytometry. Also to support the cell death identification, p38MAPK protein which is known to have role on cell death, was measured too by Western blotting (a protein identification technique) and every measurement was done at two different time intervals (at 6th and at 18th hour) from the stress exposure.
Steady increase in the percentage of dying cells was found after 18 hours from the UV exposure which indicates its usability as a positive control in our future work where as it was absent in actinomycin treatment which needs further study to increase and optimize its concentration. Both type (serum opsonised and non opsonised) yeast particle was found to decrease the mitochondrial membrane potential at 6th hour of study which was abolished at 18th hour. Since there was no result found from the annexin staining and Western blot analysis that is why we couldn’t draw any conclusion about the cell death induction by yeast particle. About the loss of mitochondrial membrane potential we extrapolated our result with similar types of study and assumed the beta glucan of yeast particle was involved here by producing reactive oxygen species via reduced form of nicotinamide adenine dinucleotide phosphate.
3. List of Contents Page NO 1. Abstract 2 2. Popular summery 3 2. List of Content 4 3. Abbreviations 4 4. Introduction 5-6 5. Aim 7
6. Materials and Methods 7-9
7. Results 9-13 8. Discussion 14 9. Acknowledgements 15 10. List of References 15-18 12. Fig1 8 13. Fig 2 10 14. Fig 3 11 15. Fig 4 12 16. Fig 5 13 4. Abbreviations
TMRE- Tetramethylrhodamine ethylester perchlotare FITC – Fluorescein isothiocyanate
MAPK- Mitogen-activated protein kinase PI – Propidium iodide
SDS PAGE- Sodium sulphate polyacrylamide gel electrophoresis PS- Phosphatidyl serine
5. Introduction 5.1. Macrophage
The macrophage is a monocyte derived cell that evolves when monocytes migrate to body tissues. It is a type of white blood cell that displays a wide array of activities such as ingesting debris, carrying antigen and presenting it to the T-cells during infection, digesting foreign organisms and tumor cells etc. Some macrophages ingest necrotic cells in particular areas such as macrophages in the lung, chuffer cells in the liver, and microglia in the neural tissue (Van Furth et al., 1972).
5.2. Apoptosis
Apoptosis is also known as programmed cell death. Apoptotic signals can be generated both from internal and external sources such as DNA damage or defects in DNA repair, cytotoxic drug exposure/irradiation, death signals. Apoptosis can be recognized by a set of biochemical and physical changes in nucleus, cytoplasm and plasma membrane (Schwartzman et al. 1993). Disruption of contact with neighbors, becoming round and shrinking are the common features of early apoptotic cells. In addition, apoptotic cells can display various signs of apoptosis e.g. chromatin densing to form aggregates, fragmentation by endonucleases, dilation of endoplasmic reticulum, forming vacuoles after swelling of cisternae in the cytoplasm, and disintegration of cell junctions in the plasma membrane leading to membrane blebbing. The nucleus becomes convoluted, turns into encapsulated fragments, and breaks apart forming apoptotic bodies. Phagocytes can recognize the apoptotic cells by their loss of sialic acids (Falasca et al., 1996) and by the asymmetry of phospholipids together with translocation of phosphatidylserin to the outer membrane (Fadokl et al., 1992).
5.3. Initiation of apoptosis (Intrinsic Pathways)
The intrinsic pathway is triggered by cytotoxic stress, which induces the translocation of proapoptotic Bcl-2 family members, such as Bax, to the mitochondria. This leads to the release of mitochondrial chromosome c in to the cytosol, where it promotes the oligomerisation of the pro apoptotic factor Apaf into a complex called the apoptosome. The apoptosome recruits and activates caspase-9 which in turn promotes the activation of caspase-3 and lead to apoptosis.
5.4. Extrinsic Pathways
The extrinsic apoptotic pathway is triggered by ligand binding to cell-surface receptors, resulting in the recruitment of various proteins to form the death-inducing signaling complex. This complex promotes activation of caspase-8, which in turn activates caspase-3. Caspase-3 then induces the cellular changes that characterize apoptosis.
5.5. Reactive Oxygen Species and Mitochondrial Dysfunction
Oxidative stress is a key factor in inducing apoptosis which is a consequence of having excess levels of free radicals in the cell. Free radicals are highly reactive molecules which are formed during different kinds of biochemical reaction take place in the cell. Many scavengers molecules also known as antioxidants which are found in the cell are engaged to eliminate these free radicals. This oxidative stress can cause damaging effect to cellular components such as cell membrane, DNA, and proteins. Several mechanisms are involved in this cell death process
through this oxidative stress including the release of apoptosis-inducing factors (Bredensen et al., 1996). Formation of excess reactive oxygen species can also lead to cell damage and death through disrupting the function of mitochondrial membrane. It is well known that the mitochondria store calcium and regulate the calcium levels in the cell which is particularly crucial in neurons. Chemical communication between neurons are maintained by this calcium level. Where as excess internal level can be toxic to neurons (Choi et al., 1995). Therefore, the ability of the mitochondria to actively sequester calcium is very important to maintain neuronal function and survival. This regulation of calcium level by mitochondria can also be interrupted by oxidative stress. Mitochondrial dysfunction can lead to both apoptosis and necrosis (Kroemer et al., 1995). Mitochondrial dysfunction is a cause of becoming whole in the membrane through which mitochondria release their contents, including calcium and a molecule called cytochrome c. Both calcium and cytochrome c can activate caspases and can play a role in apoptosis.
5.6. MAP Kinase
Mitogen-activated protein kinase (MAPK) cascades convey many signals from the cell surface to nucleus. Upon activation this MAP kinase becomes phosphorylated. Five different groups of MAP kinases have been described in mammals, p38 MAP kinase being one of them. The mostly studied vertebrate MAPKs are ERK1/2(extracellular signal-regulated kinases), JNK (Jun-N-terminal kinase), and p38 kinases. It is well known that various kinds of stimuli including stress, cytokines, mitogens etc can activate MAPK, where as JNK and p38 kinases mainly respond to stress stimuli (Pearson et al., 2001). Many extracellular stimuli can activate p38 kinase in macrophages, neutrophils, and T cells (Ono K et al., 2000). The activated p38 kinase takes part in macrophage and neutrophil functional activities like respiratory burst, chemo taxis, granular exocytosis, adherence, apoptosis, and in T-cell differentiation (Ono K et al., 2000).
5.7. Apoptosis and p38MAPK
Activation of p38MAPK pathway by a wide range of agents, such as NGF withdrawal and Fas ligation (Kummer et al., 1997;Juo P et al., 1997) and apoptosis are often connected. The cysteine proteases or the caspases play the central role, and are expressed as inactive zymogens (Henkart et al., 1996; Alnemri et al., 1996). p38 MAPK activation can be blocked by caspase inhibitors through Fas cross linking, where caspase activation has a down regulating effect (Juo P et al., 1997). Caspase activation, on the other hand, is up regulatory by the over expression of dominant active MKK6b(mitogen-activated protein kinase kinase) and hence the cause of cell death (Cardone et al., 1997;Huang et al., 1997). According to this the p38 MAPK pathway can both up and down regulate apoptosis but the mechanism is not clear. Some evidences suggests that, p38MAPK activation induces cell type and stimulus dependent apoptosis. In Jurkat T-cells, p38MAPK is activated during Fas ligation, however it is not required for apoptosis (Huang et al., 1997; Juo P et al., 1997). On the other hand, in FS-4 fibroblasts the inhibitor of p38 MAPK, SB203580 can block the apoptosis induced by sodium salicylate (Schwenger et al., 1997). In PC12 cells, apoptosis is induced by NGF withdrawal (Kummer et al., 1997), and in bovine pulmonary artery endothelial cell apoptosis can be induced by TLR-1(Yue TL et al., 1999).
6. Aim
The aim of this study was:
• To develop a method for studying apoptosis in J774 cell model. Moreover, the purpose was to identify suitable positive controls in this cell-line.
• To see the effect of yeast particles (non opsonised and opsonised by serum components) on apoptosis.
• To quantify apoptosis at different time intervals (after 6hours and 18 hours) • To correlate apoptosis with phosphorylation of p38MAPK.
7. Materials and Methods:
7.1. Reagents: Hams F-10 GLUTA-maxTM-1 medium, fetal calf serum, penicillin, and streptomycin were purchased from Life Technologies (United Kingdom), FACS FlowTMwas from
BD Biosciences, TACSTM Annexin V-FITC was from R&D system (USA), HEPES,
Mercaptoethanol and Actinomycin were from Sigma, and TMRE were purchased from Invitrogen. Sample buffer and running buffer were purchased from Bio Rad (CA). Phosphop38 MAPK antibody was obtained from Cell Signaling Technologies and p38 MAPK antibody came from New England Biolabs Technologies. ECL system and Nitrocellulose membrane was purchased from Amersham. Accutase was from Fisher Scientific. Triton X- 100 Sigma, Sodium Dodecyl Sulfate (SDS) Sigma, Sodium Deoxicholate, Monobasic sodium phosphate, NaCl were purchased from Sigma, Complete mini protease inhibitor cocktail came from Roche.
7.2. J774 Cell Culture: J774 cell are murine macrophages obtained from tumors of female
BALB/c mice. The J774 cells were cultured in F-10+GlutamaxTM-1 medium supplemented with 10% heat inactivated foetal bovine serum, penicillin(100 U/ml), and streptomycin (1000 µg/ml) and 20mM HEPES in a humidified atmosphere of 5% CO2 at 37 oC. Cells were collected by scraping at approximately 70% confluency and were seeded in 12 and 6 well plate at 0.5x10 6 cells / well for flowcytometry study, and 2x106cells/ per well for western blotting respectively, 3days before experiments. Accutase was used to collect the cell duringflow cytometry study. The cells used for western blotting experiments were collected by scrapping
7.3. Yeast Particle preparation: Heat inactivated baker’s yeast was homogenized 10 times in
KRG, washed (1000 x g, 3min, at room temperature) and diluted to 1x108/ml in KRG. The yeast were opsonised in 25% human serum, for 30min at 37o C, washed and resuspended to 3x106 /ml. Non opsonised yeast were treated in the same manner, with the exception in that no human serum was added.
7.4. Staining and Analysis of Apoptosis by Flowcytometry.
Figure : 1. A. In side the circle the cell population is
(excluding the debris) which was used to study the apoptosis. We know from the basic of cytometry that forward scatter reflect the size and shape whereas the reverse scatterreflects the internal complexity of the cell. In case of similar types of cell we will get the cell
an specific region.
And Figure B was for healthy control in our experiment. Among the four different quadrants Fig1.B, below left quadrant is negative for both FL1 and FL2 that me
as below right quadrant is positive for FL1 which representing the early ap
Quadrant of up right corner is positive for both FL1 and FL2 which representing the late apoptotic or necrotic cell.
7.4.1. Annexin-V staining: After collection by pippeting, the cells were washed once in binding
buffer (1X).The samples were incubated with FITC
for 16min on ice. The reaction was stopped by addition of cold binding buffer and the
were evaluated by flow cytometry. In healthy cell, without the translocation of phosphatidyl serine, it will not be annexin V positive and corresponding pic will be at the lower field in histogram. In order to distinguish between apoptosis and nec
counterstained with Propidium Iodide (PI) before evaluation.
7.4.2. TMRE staining: Collected cells were washed and incubated with TMRE (2µM) at 37
for 15 min and the results were evaluated by flow cytometry. In healthy cells with membrane potential, this TMRE dye will enter into the mitochondria and the corresponding
A
.4. Staining and Analysis of Apoptosis by Flowcytometry.
A. In side the circle the cell population is representing the healthy J774 cell (excluding the debris) which was used to study the apoptosis. We know from the basic of cytometry that forward scatter reflect the size and shape whereas the reverse scatterreflects the
cell. In case of similar types of cell we will get the cell
And Figure B was for healthy control in our experiment. Among the four different quadrants quadrant is negative for both FL1 and FL2 that means healthy live cell, where
is positive for FL1 which representing the early apoptotic ce
is positive for both FL1 and FL2 which representing the late
After collection by pippeting, the cells were washed once in binding buffer (1X).The samples were incubated with FITC-conjugated AnnexinV (15.5µl/ml in PBS) for 16min on ice. The reaction was stopped by addition of cold binding buffer and the
were evaluated by flow cytometry. In healthy cell, without the translocation of phosphatidyl serine, it will not be annexin V positive and corresponding pic will be at the lower field in histogram. In order to distinguish between apoptosis and necrosis the samples were counterstained with Propidium Iodide (PI) before evaluation.
Collected cells were washed and incubated with TMRE (2µM) at 37 15 min and the results were evaluated by flow cytometry. In healthy cells with membrane potential, this TMRE dye will enter into the mitochondria and the corresponding
B
representing the healthy J774 cell (excluding the debris) which was used to study the apoptosis. We know from the basic of flow-cytometry that forward scatter reflect the size and shape whereas the reverse scatterreflects the
cell. In case of similar types of cell we will get the cell population in And Figure B was for healthy control in our experiment. Among the four different quadrants in ans healthy live cell, where optotic cell number. is positive for both FL1 and FL2 which representing the late
After collection by pippeting, the cells were washed once in binding conjugated AnnexinV (15.5µl/ml in PBS) for 16min on ice. The reaction was stopped by addition of cold binding buffer and the results were evaluated by flow cytometry. In healthy cell, without the translocation of phosphatidyl serine, it will not be annexin V positive and corresponding pic will be at the lower field in rosis the samples were
Collected cells were washed and incubated with TMRE (2µM) at 37oC 15 min and the results were evaluated by flow cytometry. In healthy cells with stable membrane potential, this TMRE dye will enter into the mitochondria and the corresponding pic
will appear in higher field in the histogram, but in apoptotic cells with disrupted mitochondrial membrane potential (∆ψm), this TMRE dye will disperse throughout the cytosol and corresponding pic will shift left in the lower field in histogram, i.e. showing a decrease in the mitochondrial membrane potential. The fluorescence of individual cells stained by FITC-conjugated AnnexinV and TMRE was measured by flow cytometry, using a Becton Dickson FACSCalibur (Becton Dickinson, San Jose, CA/USA) Mean fluorescence values were determined, by counting 10,000 cells/sample. Gating was performed by using forward scatter versus side scatter (excluding cell debris). Auto fluorescence of unstained cells was routinely analyzed and subtracted from the fluorescence values of the stained cells.
7.5. Cell lyses and analysis of p38MAPKby western blotting: The J774 cells (2x106/well ) were treated with actinomycin D (0.025mg/ml) for 18 hours, UV radiation (10 min and 15 min), or with serum opsonised and non opsonised yeast particles, (1:5) for 30 min. Cells were resuspended in ice cold RIPA buffer (1% Triton X-100, 0.1% Sodium Dodecyl Sulphate(SDS), 0.5% Sodium Deoxycholate, 9.1mM monobasic sodium phosphate, 150mM NaCl, supplemented with Complete mini protease inhibitor cocktail(Roche), 1 mM Naortovanadate, and 5 µM dithiothretol ( Welin et al., 2008)) for 15 minutes. The cell were collected by scraping and centrifugation (10000 x g,10min,at 4oC), where after the lysine was diluted with sample buffer(3:1).The samples were subjected to electrophoresis and SDS-PAGE, and transferred to nitrocellulose membrane(100 V,1Hour).The membrane was blocked in TBS-5%milk for 1hour at room temperature. The membranes were incubated with a phospho-p38MAPK antibody (1:2000) for one and half hour at room temperature, followed by a secondary HRP conjugated anti rabbit antibody (1:2000). To visualize total amounts of p38MAPK proteins, the membranes were stripped and reblotted with a rabbit polyclonal anti-p38MAPK antibody (1:2000).
7.6. Statistical Analysis: All experiments were repeated at least four times and each separate
experiment was run in duplicates or triplicates. Data are presented as Mean ± SD. Data from each experiment were analyzed by Student’s t test and p< 0.05 was considered statistically significant.
8. Results:
To induce apoptosis the J774 cells were exposed to UV for two different time points (10 min and 15 min) keeping fixed distance (10cm) from the UV source. In order to find a proper positive control actinomycin D were used (0.025mg/ml) (data not shown). Two different approaches to collect the cells were used, namely Accutase (300µl/well) followed by pipeting and cell scrapping but finally we choose accutase using since this process protect the cell from mechanical disruption. Apoptosis was evaluated by flow cytometry after 6 and 18 hours.
Fig.2. Effect of actinomycin and UV radiation on apoptosis after 6 hours. J774 cells (0.5x106 /well) were grown to 70% confluency. Apoptosis was induced by UV for 10 and 15 minutesat the distance of 10 cm, or with actinomycin D (0.025mg/ml). 6 hours after the induction of apoptosis, the cells were incubated with accutase (300µl, for 30 minutes), prior to collection by vigorous pipetting. The cells were stained either by FITC conjugated annexin V(15.5µl/ml 16 minute on ice), or TMRE (2µM, 30 min at 37 o C ) where after apoptosis and mitochondrial membrane potential was evaluated by flow cytometry. Gating was performed using forward versus side scatter, counting 10000 cells per sample. None of the data was statistically significant. Data are presented as the mean ± SD of four separate experiments.UV treated cells were stained a very small percentage with TMRE after 6 hours study but not any one was statistically significant. Actinomycin also induced the mitochondrial membranepotential loss to a few percentages of macrophage but still it is statistically insignificant. Nochange was there with annaxin V staining after 6 hour study.On the other hand we find an increased numbers of cell stained by both annexin V andTMRE after 18 hour study. UV treated cells were found more apoptotic and it was statisticallysignificant for both annexin V staining and TMRE staining.
Fig.3. Effect of actinomycin and UV radiation on apoptosis after 18 hours. J774 cells (0.5x106 cells/well) were grown to 70% confluency. Apoptosis was induced by UV for 10 and 15 minutes at the distance of 10 cm, or with actinomycin (0.025mg/ml). 18 hours after induction of apoptosis, the cells were incubated with accutase (300 µl, for 30 minutes), prior to collection by vigorous pipetting. The cells were stained either by FITC conjugated annexin V (15.5µl/ml 16 minute on ice), or TMRE (2µM, 30 minute, at 37oC) where after the apoptosis and mitochondrial membrane potential was evaluated by flow cytometry. Gating was performed using forward versus side scatter, counting 10000 cells per sample. Apoptotic cell percentage both for 10 and 15 UV exposure were statistically significant compared to control ( p=0.036 and 0.027 for TMRE staining and p=0.03 and 0.007 respectively for annexin staining). Data represent the mean ± SD of four different experiments.
Fig.4. The effect of yeast on the induction of apoptosis. To study the effect of yeast particles
on apoptosis macrophages were incubated with yeast (1:5 or 1:10, NOY-non opnised yeast; SOY- serum opsonised yeast) for 1 hour. Non-bound particles were washed away, and the cells were incubated for additional five hours, prior to collection by the combination of accutase, and pipeting. As a control, cells were incubated with actinomycin (0.025mg/ml). The differences in apoptosis were statistically significant for TMRE for both serum opsonised (p=0.031), and non opsonised yeast (p= 0.05), compared to control. Data represent the mean ± SD of four separate experiments.
Fig. 5. Effect of yeast (both non opsonised and serum opsonised) on apoptosis after 18 hours
study. Macrophages were incubated with yeast both serum opsonised and non opsonised for one hour. Non bound particles were washed away and incubated 17 more hours. After that it was collected by accutase and pipeting. Actinomycin treated macrophages underwent apoptosis significantly (p=0.047) for annexin compared to control and others are statistically insignificant. Data represent the mean of four separate study ± SD.
Macrophages treated with serum opsonised yeasts, stained with TMRE showed more apoptosis (18% and 30% for 1:5 and 1:10 respectively) after 6 hours (Fig 4) but the apoptosis was reduced to 8% and 12% after 18 hours(Fig %). Non opsonised yeast induced a decrease in the mitochondrial membrane potential after 6 hours (Fig 4), but only the higher MOI displayed a mitochondrial damage 18 hours after induction (Fig 5).Similar results were obtained for non-opsonised yeasts (Fig 4 and 5).The yeast particles had no effect on the increase in annexin-V staining, independently of serum opsonisation at any time point (Fig 4 and Fig 5).
Phosphorylation of p38 MAPK in J774 macrophages. Detection of phosphorylated
p38MAPK protein after 30 min, induced by yeast particle and detected using western-blot analysis. Cells were incubated with yeast for 30 minutes, and lysed in RIPA buffer prior to collection by a cell scraper and vigorous pipetting. The supernatant was kept in -70oC. Induction of apoptosis by UV was done 10 and 15 min and incubated for 18hours. Also cells were treated with actinomycin (0.025mg/ml) for 18 hours. After that both UV and actinomycin treated cells were collected followed by lyses by RIPA buffer and vigorous pipeting. After that diluted supernatant was separated by electrophoresis and transferred to nitrocellulose membrane. The membrane was blocked with milk followed by incubation with antibodies. From left, first lane was for ladder, followed by second, third, fourth, fifth, and sixth lane was for control, actinomycin, non opsonised yeast, serum opsonised yeast, and UV respectively.
9. Discussion:
Actinomycin D- treated macrophages stained by TMRE, showed only a small increase after 6 hours (15%) and after 18 hours(16%) (Fig 2 and 3, black bars). Therefore, this concentration of Actinomycin D (0.025 mg/ml) was not suitable one as a positive control. Since it showed statistically significant increase at Fig 5 (p=0.047) but it was disappeared in other experiments. More experiments investigating the effect of other concentrations should be executed.
Exposure of UV had no effect on neither mitochondrial damage, or on the number of apoptotic cells after 6 hours (Fig 2). After 18 hours on the other hand UV induced a 15-20% increase in TMRE staining, and 45-55% increase in the number of apoptotic cells (Fig 3). Results indicating that it takes time to initiate apoptosis in J774 cell. According to the lack of induction apoptosis by yeast particles in J774 cells (fig 4), we can conclude that the yeast particles can disrupt the mitochondrial membrane potential (∆ψm).
The possible explanation of above nature is either that all apoptotic symptoms doesn’t go together or that this mitochondrial membrane potential disruption by yeast particle is not an apoptotic symptom at all, since it abolished with time. Mitochondria has control over apoptosis by generation of ATP (Leist M et al., 1997) and maintaining membrane potential and also by allowing some apoptogenic protein permeability to cytosol (Zoratti M et al., 1995; Kroemer G et al., 2000). It may raise the possibility that membrane potential loss is a cause of apoptosis induction perhaps the signal amplifier to convey further apoptotic process (Ly JD et al. 2003). In order to become confirm about this apoptosis of mouse macrophage by yeast particle, we need to do another kind of staining, such as TUNEL or we can study the morphology under microscope. After 18 hours of study we didn’t find any phosphorylated p38MAPK but after 30 min of study we found some weak bands (data not shown) which we unfortunately were not able to identify due to handling mistakes. Upon re-blotting the membrane with p38MAPK antibody the blot came out empty. This could be due to low protein content in the sample or that proteins were stripped away during re-blotting. The time-kinetics for the intracellular signal generated by yeast also need to be investigated in more detail. One possibility is that the phosphorylation of p38 MAPK has already taken place and that 18 hours on incubation no phosphorylaton of p38 can be detected. Thus no conclusion can be drawn on the activation of p38MAPK, and further experiments need to be done in future. Another possible explanation behind that empty membrane could be that the incubation with the primary antibody was too short, antibody concentration was too low, or that the antibody was too old.
Also from Fig-2 we find some loss (∆ψm) though it was not statistically significant and at 18 hour study (Fig 3) we find significant loss of mitochondrial membrane potential(∆ψm) along with other apoptosis symptom (i.e. PS exposure). Again we found that, this yeast particle induced the loss of mitochondrial potential (Fig 4) but that was abolished after 18 hour. The possible reason behind the membrane potential loss may be the production of reactive oxygen species via NADPH oxidase by yeast glucan. Study shows that yeast glucan increases the production of hydrogen-peroxide as well as phagocytosis of macrophage ( S. Brattgjerd et al., 1994). More investigation is needed to elucidate it. Also we found that, UV is good candidate to become a positive control and for actinomycin we need to optimise the concentration for J774 macrophage.
10. Acknowledgements:
I would like to express my gratitude to those that have helped in completion of this project. First I would like to thank Prof. Olle Stendahl for giving me the opportunity to work, also for his vast experiences backed encouragement. Special thanks to Veronika for her unwavering support, Anita for her teaching and love. I would also like to thank Eyler, Alex, Amanda, Daniel, Tommy, Tony, Fredric, Marie, Atem, and many of my friends for making their resources and knowledge available to me.
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